Estrogen action in reproductive tract tissue involves a mechanism which includes interaction with a receptor protein with subsequent activation and localization in the nucleus. After hormone stimulation, nuclear receptor occupancy followed a bimodal temporal pattern consistent with stimulation of certain tissue responses such as DNA synthesis or enzyme induction. The bimodal receptor pattern was evident in other responsive target tissues such as human MCF-7 cell tumors. DNA synthesis in tumors or uteri can be produced with discontinuous stimulation with a weak agonist and occurs at similar times as the two nuclear receptor events. We have purified (approximately 11000-fold) the mouse uterine estrogen receptor by steroid affinity chromatography and have characterized it by ligand affinity labeling as well as immunoblot analysis with a monoclonal antibody. Studies indicate multiple forms which are proteolytic fragments. Proteolytic processing of the receptor involves a two-step reaction. The first involving a nuclear cysteine protease and the second a soluble enzyme preliminarily characterized as a cathepsin protease. Additional biochemical characterization of the estrogen receptor protein with monoclonal antibodies demonstrated a doublet form differing by 1500 MW. This receptor form was specific to the nucleus and not found in the soluble fraction. The proportion of the doublet varied depending on the biological activity of the compound with potent agonists producing a greater amount of the upper band form. The profile of the nuclear doublet changed during estrogen stimulation in which the two components were of equal proportion until the time of the second nuclear peak at which time the proportion changed to be primarily the lower band form. Preliminary data indicate both bands are phosphorylated, the upper to a greater extent suggesting this as the protein modification for doublet formation. Experiments to investigate other processes such as acylation or glycosylation are also under study. Receptor modification may be a signal for cellular processing of the protein or programming for its interaction with specific nuclear responsive sites involved in hormone stimulation.